The genome sequence of the marbled rockcod, Notothenia rossii Richardson, 1844

We present a genome assembly from an individual Notothenia rossii (the marbled rockcod; Chordata; Actinopterygii; Perciformes; Nototheniidae). The genome sequence is 1,042.9 megabases in span. Most of the assembly is scaffolded into 12 chromosomal pseudomolecules. The mitochondrial genome has also been assembled and is 21.68 kilobases in length. Gene annotation of this assembly on Ensembl identified 24,432 protein coding genes.


Background
Notothenia rossii (marbled rockcod) (Figure 1) is a member of the notothenioid group (Suborder: Notothenioidei), a well-established fish radiation inhabiting the seas surrounding the Antarctic continent (Eastman, 2005;Matschiner et al., 2011).The species belongs to the Nototheniidae family, the largest subgroup of the notothenioids comprising 49 species (Eastman, 2005;Eastman & Eakin, 2021).The notothenioid radiation is characterised by the appearance of antifreeze genes (afgp) (Chen et al., 1997), which are present in all Antarctic members of the group (cryonotothenioids) including the Nototheniidae, and along with other adaptations enable their survival to extreme cold conditions of the Southern Ocean.
The position of N. rossii populations in the water column changes during their life cycle.Adults are bentho-pelagic on the South Georgia shelf and produce large (4-5 mm) planktonic eggs in winter (Kock et al., 2004).The eggs hatch as planktonic larvae (14-30 mm TL) that develop into a blue pelagic phase (30-70 mm TL) which occupy near-surface waters.The blue phase settle in coastal kelp habitats, where they become demersal and develop a brown colouration (Burchet, 1983).Juveniles remain in coastal habitats for around five years, until they approach maturity and migrate off shore (Burchet, 1983;Hollyman et al., 2021), a distribution which also resembles another mostly sympatric species the black rockcod (Notothenia coriiceps) (Calì et al., 2017).Observed maximum body size for the marbled rockcod is usually close to 80 cm TL (Hollyman et al., 2021) with maximum reported size reaching 92 cm TL (DeWitt et al., 1990).Males mature earlier and spawn at smaller body size compared to females (Hollyman et al., 2021).Feeding behaviour includes a large variety of prey making this species a generalist feeder, with prey including many groups such as various fish, and crustaceans like hyperiids, mysids, amphipods, and euphausiids (krill) (Hollyman et al., 2021).
Historically N. rossii was the first major target of the fishing industry in the Southern Ocean which heavily exploited its populations, eventually leading to a ban on targeted fishing for N. rossii in 1985, when the Commission for the Conservation of Antarctic Marine Living Resources (CCAMLR) applied conservation measures for its protection (Kock et al., 2004).Recovery of fish populations after overfishing events can be influenced by oceanographic, life history traits, and connectivity, with N. rossii populations still struggling to recover.Though there is a high degree of population connectivity, the poor retention of egg and larval stages during planktonic phases, and increased mortality due to predator pressure (fur seals) potentially hinders recovery of N. rossii populations (Young et al., 2012).
Here we present a genome assembly generated using a juvenile specimen collected using a Neuston net from the Antarctic Polar Front area, during the JR19001 cruise onboard the James Clark Ross research vessel.This chromosomal assembly of N. rossii, provides an improvement on our previous assembly for this species (fNotRos1, PRJEB53175, GCA_943590865.1 (Bista et al., 2023)).

Genome sequence report
The genome was sequenced from a Notothenia rossii collected from the Southern Ocean (-54.42, -45.88).A total of 24-fold coverage in Pacific Biosciences single-molecule HiFi long reads was generated.Primary assembly contigs were scaffolded with chromosome conformation Hi-C data.Manual assembly curation corrected 58 missing joins or mis-joins and removed 4 haplotypic duplications, reducing the scaffold number by 2.58%, and increasing the scaffold N50 by 99.18%.
The final assembly has a total length of 1,042.9Mb in 942 sequence scaffolds with a scaffold N50 of 89.7 Mb (Table 1).The snail plot in Figure 2 provides a summary of the assembly statistics, while the distribution of assembly scaffolds on GC proportion and coverage is shown in Figure 3.The cumulative assembly plot in Figure 4 shows curves for subsets of scaffolds assigned to different phyla.Most (93.47%) of the assembly sequence was assigned to 12 chromosomal-level scaffolds.Chromosome-scale scaffolds confirmed by the Hi-C data are named in order of size (Figure 5; Table 2).While not fully phased, the assembly deposited is of one haplotype.Contigs corresponding to the second haplotype have also been deposited.The mitochondrial genome was also assembled and can be found as a contig within the multifasta file of the genome submission.The estimated Quality Value (QV) of the final assembly is 53.7 with k-mer completeness of 99.98%, and the assembly has a BUSCO v5.3.2 completeness of 94.9% (single = 94.1%,duplicated = 0.8%), using the actinopterygii_odb10 reference set (n = 3,640).

Sample acquisition and nucleic acid extraction
A juvenile specimen of Notothenia rossii was collected (BioSample: SAMEA12815441, fNotRos5) from the area of the Antarctic Polar Front near S. Georgia on 16 December 2019.The individual was captured using a Neuston net and preserved in a tube in the a -80°C freezer until processing.The specimen was collected by Iliana Bista and identified by Martin Collins.
The workflow for high molecular weight (HMW) DNA extraction at the Wellcome Sanger Institute (WSI) includes a sequence of core procedures: sample preparation; sample      et al., 2013) and uses these annotations to select the final mitochondrial contig and to ensure the general quality of the sequence.
A Hi-C map for the final assembly was produced using bwa-mem2 (Vasimuddin et al., 2019) in the Cooler file format (Abdennur & Mirny, 2020).To assess the assembly metrics, the k-mer completeness and QV consensus quality values were calculated in Merqury (Rhie et al., 2020).

Genome annotation
The Ensembl Genebuild annotation system (Aken et al., 2016) was used to generate annotation for the Notothenia rossii assembly (GCA_949606895.1) in Ensembl Rapid Release at the EBI.Annotation was created primarily through alignment of transcriptomic data to the genome, with gap filling via protein-to-genome alignments of a select set of proteins from UniProt (UniProt Consortium, 2019).

Wellcome Sanger Institute -Legal and Governance
The materials that have contributed to this genome note have been supplied by a Tree of Life collaborator.The Wellcome Sanger Institute employs a process whereby due diligence is carried out proportionate to the nature of the materials themselves, and the circumstances under which they have been/are to be collected and provided for use.
The purpose of this is to address and mitigate any potential legal and/or ethical implications of receipt and use of the materials as part of the research project, and to ensure that in doing so we align with best practice wherever possible.
The overarching areas of consideration are: • Ethical review of provenance and sourcing of the material

Is the rationale for creating the dataset(s) clearly described? Yes
Are the protocols appropriate and is the work technically sound?Yes

Are the datasets clearly presented in a useable and accessible format? Yes
Competing Interests: No competing interests were disclosed.

Reviewer Expertise: Genomics
I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

Chenhong Li
Shanghai Universities Key Laboratory of Marine Animal Taxonomy and Evolution, Shanghai Ocean University, Shanghai, China The work is well done.Data collected and assembly and annotation methods all followed lasted high standard.Introduction is well writen.I only have two suggestions: add a phylogenetic tree showing the relationship of the group and relatives and note which ones have genomes available.
This may suggests what need to be done to fill the gaps; comparison the statistics between the current genome assembly and the previous one.

Is the rationale for creating the dataset(s) clearly described? Yes
Are the protocols appropriate and is the work technically sound?Yes

Are sufficient details of methods and materials provided to allow replication by others? Yes
Are the datasets clearly presented in a useable and accessible format?Yes Competing Interests: No competing interests were disclosed.
Reviewer Expertise: Phylogenetics, comparative genetics I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.The manuscript titled "The genome sequence of the marbled rockcod, Notothenia rossii Richardson, 1844" presents a comprehensive genome assembly and annotation of the marbled rockcod, Notothenia rossii.The assembly spans 1,042.9megabases and includes 12 chromosomal pseudomolecules, along with the mitochondrial genome.Gene annotation on Ensembl identified a total of 24,432 protein-coding genes.
The manuscript presents a significant advancement in the genomics data of Notothenia rossii, offering a comprehensive genome assembly and annotation.It serves as a critical resource for advancing research in comparative genomics and evolutionary biology within Perciformes.To enhance its scientific impact, I recommend the authors consider incorporating additional comparative analyses. Comments:

INTRODUCTION
The introduction lacks coherence and flow.It transitions abruptly from general fish biology to mentioning antifreeze genes (afgp) without clear context.Subsequently, it briefly touches on the fishery importance and overexploitation issues, but fails to articulate the specific goals of obtaining the genome.What studies are envisioned with an available genome?Why sequence a higher-quality genome than previously achieved?What new insights can be gleaned at the chromosomal level that were not possible before?Additionally, the contribution of the mitochondrial genome is not clearly articulated.
The manuscript would benefit significantly from a more structured development and utilization of the sequenced genome.If the aim is to study the antifreeze genes (afgp), comparative bioinformatics work with similar genes from other species should be included.For conservation studies, numerous comparative analyses could be conducted, which are not explored in the current manuscript.There is a clear need to outline the rationale behind obtaining a high-quality genome for this species.

METHODOLOGY
The manuscript lacks crucial details regarding the methods for sampling and preserving fish specimens after sacrifice.Specifically, there is no information provided on how samples were taken and preserved for subsequent analyses.Furthermore, it is unclear whether the study received approval from an ethics committee.If an ethics committee approval was obtained, details regarding which committee granted approval (e.g., Institutional Animal Care and Use Committee, IACUC) should be included for transparency and compliance with ethical standards.

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The manuscript mentions a previous sequencing effort in the species but fails to discuss the sequencing methodologies employed or highlight any advancements or differences in sequencing technologies used in the current study.

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It is crucial to address why individuals of both sexes have not been sequenced.This omission is particularly significant because sequencing both sexes could reveal sex chromosomes, sex-determining genes, or allow for a more comprehensive integration of genomic data.

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Understanding the genomic basis of sex determination and potential sex-specific differences is essential for a thorough understanding of the species.Integrating data from both male and female individuals could provide insights into sex-linked genomic regions and contribute to broader evolutionary and biological studies.Therefore, I recommend the authors address these points to strengthen the manuscript's comprehensiveness and relevance to the field of genomics and evolutionary biology.

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The manuscript lacks essential information regarding the RNA extraction process, specifically which tissue(s) were utilized.It is crucial to clarify why these particular tissues were selected for RNA extraction.

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To enhance the manuscript's clarity and methodological transparency, it is recommended that the authors provide a detailed explanation of the tissues chosen for RNA extraction, including any biological or experimental justifications for their selection.This addition will strengthen the scientific rigor of the study and improve its reproducibility by facilitating a better understanding of the experimental design.

RESULTS
Figures 3, 4, and 5 in the manuscript depict sequence quality and assembly metrics; however, there is limited subsequent analysis or discussion regarding the insights gained from this information.These figures are critical for evaluating the reliability and completeness of the genome assembly, but their potential to inform subsequent results or biological implications remains underexplored.

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To strengthen the manuscript, it is advisable to leverage the data presented in these figures to provide deeper insights into the genome's characteristics.For instance, discussions could include how assembly quality influences the identification of specific genomic features, comparative genomic analyses, or evolutionary implications within the species or related taxa.Additionally, linking these metrics with functional genomic data or highlighting specific genomic regions of interest would enhance the manuscript's scientific impact and relevance.

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Addressing these points will ensure that the figures not only serve as quality assessments but also contribute substantively to the overall findings and interpretations presented in the manuscript.
The study presents a genome assembly of the marbled rockcod, Notothenia rossii, spanning 1,042.9megabases with scaffolded chromosomal pseudomolecules and a fully assembled mitochondrial genome of 21.68 kilobases.Gene annotation identified 24,432 protein-coding genes on Ensembl.While this assembly represents a comprehensive genomic dataset for Notothenia rossii, the manuscript falls short in leveraging this resource for broader comparative biological insights.

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To strengthen the manuscript, the authors should consider discussing potential applications of the genome assembly beyond basic gene annotation.Highlighting specific research questions that can be addressed with this dataset, such as adaptations to cold environments or evolutionary trajectories within Antarctic fish, would broaden its significance and appeal to a wider audience in the scientific community.
Is the rationale for creating the dataset(s) clearly described?Partly Reviewer Expertise: Molecular Biology.Fish reproduction and Endocrinology.Genomics.Computational Biology I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

Figure 1 .
Figure 1.Photograph of Notothenia rossii from the British Antarctic Survey (not the specimen used for genome sequencing).

Figure 2 .
Figure 2. Genome assembly of Notothenia rossii, fNotRos5.1:metrics.The BlobToolKit snail plot shows N50 metrics and BUSCO gene completeness.The main plot is divided into 1,000 size-ordered bins around the circumference with each bin representing 0.1% of the 1,042,906,029 bp assembly.The distribution of scaffold lengths is shown in dark grey with the plot radius scaled to the longest scaffold present in the assembly (97,391,511 bp, shown in red).Orange and pale-orange arcs show the N50 and N90 scaffold lengths (89,684,120 and 60,281,083 bp), respectively.The pale grey spiral shows the cumulative scaffold count on a log scale with white scale lines showing successive orders of magnitude.The blue and pale-blue area around the outside of the plot shows the distribution of GC, AT and N percentages in the same bins as the inner plot.A summary of complete, fragmented, duplicated and missing BUSCO genes in the actinopterygii_odb10 set is shown in the top right.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/fNotRos5_1/dataset/fNotRos5_1/snail.

Figure 3 .
Figure 3. Genome assembly of Notothenia rossii, fNotRos5.1:BlobToolKit GC-coverage plot.Sequences are coloured by phylum.Circles are sized in proportion to sequence length.Histograms show the distribution of sequence length sum along each axis.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/fNotRos5_1/dataset/fNotRos5_1/blob.

Figure 4 .
Figure 4. Genome assembly of Notothenia rossii, fNotRos5.1:BlobToolKit cumulative sequence plot.The grey line shows cumulative length for all sequences.Coloured lines show cumulative lengths of sequences assigned to each phylum using the buscogenes taxrule.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/fNotRos5_1/dataset/fNotRos5_1/cumulative.
size distribution was evaluated by running the sample on the FemtoPulse system.RNA was extracted from tissue of fNotRos5 in the Tree of Life Laboratory at the WSI using the RNA Extraction: Automated MagMax™ mirVana protocol(do Amaral  et al., 2023).The RNA concentration was assessed using a Nanodrop spectrophotometer and a Qubit Fluorometer using the Qubit RNA Broad-Range Assay kit.Analysis of the integrity of the RNA was done using the Agilent RNA 6000 Pico Kit and Eukaryotic Total RNA assay.Protocols developed by the WSI Tree of Life laboratory are publicly available on protocols.io(Dentonet al., 2023).SequencingPacific Biosciences HiFi circular consensus DNA sequencing libraries were constructed according to the manufacturers' instructions.Poly(A) RNA-Seq libraries were constructed using the NEB Ultra II RNA Library Prep kit.DNA and RNA sequencing was performed by the Scientific Operations core at the WSI on Pacific Biosciences SEQUEL II (HiFi) and Illumina NovaSeq 6000 (RNA-Seq) instruments.

Figure 5 .
Figure 5. Genome assembly of Notothenia rossii, fNotRos5.1:Hi-C contact map of the fNotRos5.1 assembly, visualised using HiGlass.Chromosomes are shown in order of size from left to right and top to bottom.An interactive version of this figure may be viewed at https://genome-note-higlass.tol.sanger.ac.uk/l/?d=Fz2tF8wBS-qgF_W_5Ixaqw.

Reviewer Report 23
July 2024 https://doi.org/10.21956/wellcomeopenres.23525.r86179© 2024 Li C.This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Are the protocols appropriate and is the work technically sound?Partly Are sufficient details of methods and materials provided to allow replication by others?Partly Are the datasets clearly presented in a useable and accessible format?Partly Competing Interests: No competing interests were disclosed.

Table 2 . Chromosomal pseudomolecules in the genome assembly of Notothenia rossii, fNotRos5. INSDC accession Chromosome Length (Mb) GC%
Hi-C data were also generated from tissue of fNotRos5 using the Arima2 kit and sequenced on the Illumina NovaSeq 6000 instrument.